We broadly investigate the mechanisms used in cells to regulate gene expression at the translational level. Current research is focused on the question of how ribosomes are dissociated from mRNAs (recycled) following the completion of translation at stop codons or after irreversible stalling events in the middle of coding sequences. Without this recycling process, ribosomes would accumulate on mRNAs, limiting the cell's ability to make new protein or allowing translation of sequences that encode toxic proteins. Ribosome recycling occurs when the ATPase Rli1 (ABCE1 in higher eukaryotes) separates the 60S subunit from the mRNA-bound 40S ribosome. In prior work, we established that lack of Rli1 in the cell leads to a surprising accumulation of ribosomes in the 3'UTR and the translation of short open reading frames. We also showed that recycling of the 40S subunit in yeast requires the activity of the proteins Tma64, Tma20, and Tma22 (eIF2D, MCT-1, and DENR in mammals). Similar to loss of Rli1, we found that loss of these factors causes ribosomes to enter 3'UTRs and reinitiate new translation. The recycling factor ABCE1 is known to be upregulated in many types of cancer, suggesting that ribosome recycling is critical in cancer cells, potentially as a way to ensure an adequate supply of recycled ribosomes for new rounds of translation during rapid proliferation. We believe this process is also critical during the innate immune response because the activity of ABCE1 appears to be modulated by RNase L, a gene that is activated when the cell detects double-stranded RNA in the cytoplasm. Mutation of DENR has been shown to be associated with autism and overexpression of MCT-1 is a lymphoma driver in humans. A better understanding of the mechanism of ribosome recycling is therefore important for addressing challenges to human health. The lab primarily employs high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We also use an array of biochemical approaches, such as western blots and reporter assays, to complement this work. Finally, we are developing tools for imaging single polysomes in living yeast and mammalian cells, using fluorescence microscopy and reporters consisting of arrayed GFP molecules (SunTag). We recently investigated the role of the protein Hcr1 in yeast (eIF3j in mammals) for its role in ribosome recycling. Hcr1 is known to promote initiation of translation in 5'UTRs, but its function in recycling was less clear, with studies suggesting roles in termination or either 40S or 60S recycling. We used ribosome profiling and reporter assays to determine whether Hcr1 facilitates removal of the 60S ribosomal subunit from the mRNA-bound 40S subunit or the 40S ribosome from the mRNA. Our data clearly reveal that Hcr1 promotes 60S subunit recycling because the yeast strain missing this factor is a close phenocopy of the Rli1-depleted strain. Moreover, we did not observe a requirement for an AUG codon during 3'UTR reinitiation events. Hcr1 likely works to enhance Rli1 ATPase activity or target Rli1 to ribosomes. Intriguingly, we also found that loss of Hcr1 triggers increased expression of the RLI1 mRNA, suggesting the cell actively senses ribosome recycling fidelity and tunes Rli1 levels to ensure sufficient levels of recycling. We are also investigating the functional roles for 3'UTR ribosomes and the possibility that loss of efficient recycling under stressful environments can alter fitness. We are examining the effects of nutrient deprivation (yeast) and the innate immune response (human cell lines), for example, in reducing the efficiency of termination and recycling. Our ribosome profiling results now suggest many stresses induce translation of 3'UTRs but that the mechanism involved under glucose loss in yeast is highly specific for 40S reinitiation. During the innate immune response, activation of RNase L is thought to lead to binding with ABCE1. We are now measuring recycling efficiency in the presence of activated RNase L and have observed robust loss in recycling efficiency across multiple cell lines. Our results suggest a specific mechanism of modulating recycling when the host is infected by viruses. We have also begun to employ a new form of ribosome profiling by footprinting the 40S (as opposed to 80S) ribosome. Data from this experiment now directly demonstrate that loss of Tma64, Tma20, and Tma22 leads to widespread accumulation of 40S ribosomes on stop codons. We therefore conclude that the 40S ribosomes that accumulate in their absence are competent to reinitiate translation in 3'UTRs by multiple mechanisms. Using this approach, we have also shown that Tma64/eIF2D only carries out a small proportion of the 40S recycling burden. Preliminary studies in mammalian cells support this conclusion and suggest a role in controlling initiation during stress responses. We also found that the autism-associated mutants of DENR lead to clear recycling defects when introduced into yeast, thereby linking ribosome recycling failure with neurological defects. In addition, we have developed a tool to reveal translation of non-canonical ORFs. Our observation of accumulation of 40S ribosomes on stop codons in a strain lacking 40S recycling factors can serve as a signal of non-canonical termination events across the transcriptome. Surprisingly, we have found widespread evidence of such events internal to coding sequences. We believe such events are indicative of leaky scanning, where the 40S ribosome fails to find the main AUG start codon and instead initiates translation downstream, and in some cases, in a different reading frame. Initial analysis suggests such events can trigger degradation of the mRNA via the nonsense mediated decay pathway (NMD, since most out of frame ORFs end with termination codons near the 5' end of the gene). We anticipate that this technique will be useful for detection of other cases of non-canonical translation events, particularly translation of rare transcript isoforms that encode premature termination codons and are therefore subject to NMD. Finally, we have enhanced another variant of ribosome profiling that I originally introduced for studying collisions between two ribosomes. By purifying footprints protected by two ribosomes joined together (disomes), we have globally identified sites where ribosomes collide across the transcriptome. Intriguingly, we have found that all disomes are recognized the ribosome quality control pathway (RQC). It has been shown that RQC specifically recognizes disomes, leading to ubiquination by Hel2 (yeast) or ZNF598 (mammals) and subsequent degradation of the nascent peptide and the mRNA. Loss of this pathway leads to the production of toxic peptides and neurodegeneration. Our data revealed that loss of Hel2 reduces disome formation, establishing that this key quality-control pathway is widespread and does not distinguish between collided ribosomes formed on different stall-inducing motifs. Such events on apparently normal mRNA transcripts may be critical for ensuring the synthesis of high-quality protein.